Definition of the bacterial N-glycosylation site consensus sequence

The Campylobacter jejuni pgl locus encodes an N ‐linked protein glycosylation machinery that can be functionally transferred into Escherichia coli . In this system, we analyzed the elements in the C. jejuni N ‐glycoprotein AcrA required for accepting an N ‐glycan. We found that the eukaryotic primary consensus sequence for N ‐glycosylation is N terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N ‐glycosylated when they were either artificially introduced or when they were present in non‐ C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N ‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in C. jejuni N ‐glycoproteins. N ‐glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the −2 position. Thus, bacterial N ‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.